Adhesion receptor-ligand interactions influence vascularized organ allografts at multiple levels, including trafficking of host leukocytes into the graft, costimulation of alloreactive T cells through leukocyte integrins, or directed killing of donor target cells by conjugate formation with cytotoxic, allospecific effector cells. We have demonstrated that, upon engagement, lymphocyte function-associated antigen-1 (LFA-1,aL2, CD11a/CD18) generates signals leading to stabilization of Th1 cytokine transcripts including those encoding IFN-?, TNF-a and IL-2. We have shown that this T cell mRNA stabilization is directed at modulation of transcript degradation through target AU-rich sequences, and requires LFA-1 engagement-induced nuclear-to-cytoplasmic translocation of the RNA binding protein HuR. We have also demonstrated that LFA-1-triggered, PI3-kinase- dependent activation of the guanine nucleotide exchange factor Vav-1 is involved, consequently activating the Rho family GTPases Rac1 and 2 which, through as yet uncharacterized signaling mechanisms, promote stabilization of highly labile transcripts. These events coincide with rapid, LFA-1-induced protein-protein interactions involving HuR and members of the heterogeneous nuclear ribonucleoprotein (hnRNP) family. These findings have led to our overall hypothesis that engagement of T cell integrin adhesion receptors confers coactivatory properties which, through MAP kinase (MAPK) cascades and modulation of the mRNA binding protein HuR, result in stabilization of labile transcripts encoding vascular pathology-inducing cytokines, leading to acute and chronic rejection of vascularized allografts. Specific proposals now include to: (1) further define signaling components of T cell LFA-1-mediated HuR translocation and mRNA stabilization, focusing on the Rac downstream targets p38 MAPK, MAPK-activated protein kinase 2 (MK2) and novel, T cell-specific MK2 targets, using pharmacologic inhibitors, RNA interference (RNAi)-based approaches, T cells from relevant gene-deleted mice, and screens for T cell Hsp27 homologues; (2) determine the molecular features and importance of the T cell LFA-1-induced interactions between HuR and hnRNP-C, using pull-down methods, fluorescence resonance energy transfer (FRET) assays, and RNAi-based approaches; and (3) definitively evaluate the role of HuR in coactivator-dependent T cell activation, using T cells obtained from T cell-specific HuR gene-deleted mice in LFA-1-induced mRNA cytokine stabilization assays, and these T cell HuR-/- mice as recipients of cardiac and aortic allografts. The effect of the T cell HuR deletion on the vascular remodeling observed in chronic allograft rejection will be assessed morphometrically in the aortic allograft model. In vitro data will direct the design of substrate-mimetic and protein interaction site cell permeable peptides to inhibit mRNA stabilization, and to be used in vivo. This work will provide insight into cellular and molecular triggers resulting in effector cytokine production, and will direct strategies for modulating a master regulatory switch in allograft rejection, as well as other pathologies directed at the vasculature. PUBLIC HEALTH RELEVANCE: The immune system can greatly influence vascular disease, including in the development of atherosclerosis and during the rejection of transplanted organs. This work seeks to define ways in which to finely regulate those immune responses at the gene expression level, with an eye toward generating novel therapeutic agents to suppress the production of immune mediators which contribute to vascular disease.